Recombinant human acidic fibroblast growth factor gel and its preparation process

Abstract

This recombinant human acidic fibroblast growth factor (aFGF) gel contains a gel matrix consisting of one or more of sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, sodium polypropylene, sodium hyaluronate, polyethylene glycol, and gelatin; a thickener consisting of polyvinyl alcohol; and a protective agent consisting of histidine and low-molecular-weight sodium heparin. The combination of the gel matrix and the thickener polyvinyl alcohol, along with the use of histidine and low-molecular-weight sodium heparin as protein protective agents, synergistically improves the stability of the recombinant human acidic fibroblast growth factor (aFGF) gel. Research has shown that the combination of histidine and sodium heparin can be used as a protein protective agent to replace human serum albumin, providing high protective efficacy even at low concentrations and effectively reducing production costs.

Description

[Technical Field]

[0001] The present invention relates to the technical field of drug formulations, particularly to a recombinant human acidic fibroblast growth factor gel and its preparation process. [Background technology]

[0002] Acidic fibroblast growth factor (aFGF) is a multifunctional cell growth factor derived from mesodermal and neuroectodermal cells. It is a trace active substance with broad mitogenic activity and is distributed mainly in organs and tissues such as the brain, pituitary gland, nervous tissue, retina, adrenal glands, heart, and bone. Its content in other tissues is very low, and it is present at very low concentrations in serum and body fluids. As an endogenous active factor closely related to the wound healing process, aFGF has significant promoting effects on tissue wound healing, nerve damage repair, and vascular regeneration. It is used to promote wound healing in deep second-degree burns and chronic ulcers (including posttraumatic residual wounds, diabetic ulcers, vascular ulcers, and pressure ulcers).

[0003] Common dosage forms of acidic fibroblast growth factor (aFGF) include lyophilized powder, eye drops, and gel. Lyophilized powder can be used by pouring the solvent included in the packaging into a bottle containing rhaFGF lyophilized powder and then covering (engaging) the spray head included in the packaging. When used, the drug solution can be sprayed directly onto the wound after debridement. Eye drops and gel can be used directly. Gels combine the properties of polymeric materials to effectively extend the residence time of the added active factor at a specific site, improving drug absorption and bioavailability. Furthermore, their targeting function and slow drug release can reduce the frequency of clinical administration and facilitate application, making them widely competitive in the market.

[0004] Gels have the advantages of being easy to apply, pleasant to use, non-greasy, easy to wash off, able to absorb fluids exuded from tissues, not interfering with normal skin physiological functions, and having a certain moisture-retaining function, which can promote transdermal drug absorption. However, aqueous acidic fibroblast growth factor (aFGF) gels are sensitive to external conditions such as temperature, and are prone to denaturation and inactivation if left at room temperature for a long time. To address this technical issue, Chinese Patent Application No. 202111189805.1 uses carbomer 940 as the gel matrix and human serum albumin, heparin sodium, etc. as protectants to obtain a recombinant human acidic fibroblast growth factor (aFGF) gel that is not sensitive to temperature conditions. While this patent application solves the problem of low stability of aqueous acidic fibroblast growth factor (aFGF) gels, the use of large amounts of protectants inevitably increases the cost of the product. Summary of the Invention

[0005] In response to the shortcomings of the prior art, the present invention provides a novel recombinant human acidic fibroblast growth factor (aFGF) gel formulation to solve the problems in the prior art of low product stability and high manufacturing costs.

[0006] Specifically, the present invention provides a recombinant human acidic fibroblast growth factor (aFGF) gel comprising a gel matrix which is one or more selected from sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, sodium polypropyleneate, sodium hyaluronate, polyethylene glycol, and gelatin, a thickener selected from polyvinyl alcohol, and a protective agent selected from a combination of histidine and sodium heparin.

[0007] Furthermore, the present invention provides a recombinant human acidic fibroblast growth factor (aFGF) gel, in which the protective agent is selected from a combination of histidine and sodium heparin, and the sodium heparin is selected from low-molecular-weight sodium heparin, preferably having an average molecular weight of 3,000 to 8,000.

[0008] Furthermore, the present invention provides a recombinant human acidic fibroblast growth factor (aFGF) gel, wherein the mass of the histidine is 0.8 to 2‰, preferably 1 to 1.6‰, of the mass of the gel, and the mass of the heparin sodium is 0.05 to 1‰, preferably 0.08 to 0.2‰, of the mass of the gel.

[0009] Furthermore, the present invention provides a recombinant human acidic fibroblast growth factor (aFGF) gel, wherein the mass of the gel matrix is ​​0.5 to 5%, preferably 1 to 3%, of the mass of the gel.

[0010] Furthermore, the present invention provides a recombinant human acidic fibroblast growth factor (aFGF) gel, wherein the mass of the thickener is 0.5 to 3%, preferably 1 to 2%, of the mass of the gel.

[0011] Furthermore, the present invention provides a recombinant human acidic fibroblast growth factor (aFGF) gel further comprising a buffer solution, wherein the buffer salt is selected from a phosphate buffer system, a citrate buffer system, and a Tris buffer system, preferably a phosphate buffer system, and the buffer salt preferably has a pH of 6.0 to 7.0 and a concentration of 0.01 to 0.05 mol / L, preferably 0.02 mol / L.

[0012] Furthermore, the present invention provides a method for producing a gel containing recombinant human acidic fibroblast growth factor (RAF) in a relative amount of 2 to 8 × 10 3 A recombinant human acidic fibroblast growth factor (aFGF) gel containing 1.1 IU / g of aFGF is provided.

[0013] Furthermore, the present invention provides a recombinant human acidic fibroblast growth factor (aFGF) gel, which further comprises a moisturizer and a preservative, preferably the moisturizer is selected from glycerol, the preservative is selected from parahydroxybenzoic acid ester preservatives, preferably the preservative is one or more selected from methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and isopropyl parahydroxybenzoate, and the moisturizer also acts as a co-solvent.

[0014] Furthermore, the present invention provides a recombinant human acidic fibroblast growth factor (aFGF) gel, wherein the mass of the preservative is 0.1 to 3% of the mass of the gel, preferably 0.2 to 1% of the mass of the gel, and the mass of the moisturizer is 0.2 to 4%, preferably 0.5 to 2%, of the mass of the gel.

[0015] The present invention has the following beneficial effects.

[0016] In the present invention, a combination of a gel matrix and a thickener, polyvinyl alcohol, is selected, and histidine and sodium heparin are selected as protein protectants. These auxiliary materials synergistically improve the stability of the recombinant human acidic fibroblast growth factor (aFGF) gel, and the resulting gel product maintains high stability and relative activity even under high temperature conditions.

[0017] Through screening, it was found that the combination of histidine and sodium heparin can be used as a protein protective agent that can replace human serum albumin, and can achieve high protective effect even at low concentrations, effectively reducing production costs. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail and specifically below with reference to specific examples in order to better understand the present invention, but the following examples are not intended to limit the scope of the present invention.

[0019] The phosphate buffer solution described in this invention is a NaHPO, NaHPO·H0 buffer solution with a concentration of 0.02 mol / L and a pH of 6.0-7.0. The nominal amount of the reagent and preparation of the formulation are carried out under aseptic conditions. The average molecular weight of the low molecular weight sodium heparin in this embodiment of the invention is 3000-8000.

[0020] Example 1: Taking the preparation of 100g of recombinant human acidic fibroblast growth factor (aFGF) gel as an example, the specific ingredients and preparation method are as follows:

[0021] rhaFGF 4×10 5 IU Sodium carboxymethylcellulose 2g Polyvinyl alcohol 1g Histidine 0.1g Low molecular weight heparin sodium 0.02g Ethyl parahydroxybenzoate 0.05g 2g glycerol 0.189g NaH2PO4·H2O 0.089g Na2HPO4

[0022] (1) Approximately 40 ml of water for injection was taken, and preservatives and humectants in the amounts prescribed were added. The mixture was heated to dissolve, and then a gel matrix in the amount prescribed was added. The mixture was allowed to swell overnight, and then sterilized under high pressure at 121°C for 30 minutes. The mixture was then allowed to cool to room temperature, and solution A was obtained and ready for use.

[0023] (2) The ingredients of NaHPO, NaHPO·HO, thickener, and protectant were added to 50 ml of water for injection, stirred to dissolve, and then the ingredients of recombinant human acidic fibroblast growth factor stock solution were added and mixed thoroughly to obtain Solution B, which was ready for use.

[0024] (3) Solution B was filtered through a 0.22 μm filter membrane to sterilize it, and then added to Solution A. The remaining amount of water for injection was added to bring the total weight to 100 g, and the mixture was thoroughly stirred and allowed to stand to obtain a rhaFGF gel.

[0025] Example 2: Taking the preparation of 100g of recombinant human acidic fibroblast growth factor (aFGF) gel as an example, the specific ingredients are as follows:

[0026] rhaFGF 6×10 5 IU Hydroxypropyl methylcellulose 2.5g Polyvinyl alcohol 1g Histidine 0.1g Low molecular weight heparin sodium 0.02g Methyl parahydroxybenzoate 0.05g 2g glycerol 0.189g NaH2PO4·H2O 0.089g Na2HPO4

[0027] See Example 1 for the preparation method.

[0028] Example 3: Taking the preparation of 100g of recombinant human acidic fibroblast growth factor (aFGF) gel as an example, the specific ingredients are as follows:

[0029] rhaFGF 4×10 5 IU Sodium alginate 4g Polyvinyl alcohol 1.5g Histidine 0.1g Low molecular weight heparin sodium 0.02g Isopropyl parahydroxybenzoate 0.05g 2g glycerol 0.189g NaH2PO4·H2O 0.089g Na2HPO4

[0030] Comparative Example 1: Taking the preparation of 100 g of recombinant human acidic fibroblast growth factor (aFGF) gel as an example, the specific ingredients and preparation method are as follows:

[0031] rhaFGF 4×10 5 IU Sodium carboxymethylcellulose 2g Histidine 0.1g Low molecular weight heparin sodium 0.02g Ethyl parahydroxybenzoate 0.05g 2g glycerol 0.189g NaH2PO4·H2O 0.089g Na2HPO4

[0032] See Example 1 for the preparation method.

[0033] Comparative Example 2: Taking the preparation of 100 g of recombinant human acidic fibroblast growth factor (aFGF) gel as an example, the specific ingredients and preparation method are as follows:

[0034] rhaFGF 4×10 5 IU Sodium carboxymethylcellulose 2g Polyvinyl alcohol 1g Low molecular weight heparin sodium 0.02g Ethyl parahydroxybenzoate 0.05g 2g glycerol 0.189g NaH2PO4·H2O 0.089g Na2HPO4 See Example 1 for the preparation method.

[0035] Comparative Example 3: Taking the preparation of 100 g of recombinant human acidic fibroblast growth factor (aFGF) gel as an example, the specific ingredients and preparation method are as follows:

[0036] rhaFGF 4×10 5 IU Sodium carboxymethylcellulose 2g Polyvinyl alcohol 1g Histidine 0.1g Ethyl parahydroxybenzoate 0.02g 2g glycerol 0.189g NaH2PO4·H2O 0.089g Na2HPO4

[0037] The prepared rhAFGF gel was verified by the following experiments, and the test results are shown in Table 1 below.

[0038] (1) Stability test of rhAFGF gel Five grams of the gels of Examples 1 to 3 and Comparative Examples 1 to 3 were taken, sealed in aluminum tubes, and stored airtight at 25°C and 40°C, respectively. The appearance of the gels was inspected periodically, and samples were taken to measure the activity of rh-aFGF in the gels, and the relative activity value was calculated.

[0039] (2) rhAFGF activity measurement method (MTT method) After reconstitution of the working standard endotoxin for rhAFGF activity measurement, the solution was diluted 16-fold three times with the assay medium, then diluted 4-fold seven times, and then loaded with eight dilution gradients to prepare two duplicate wells. The test substance was reconstituted in the indicated amount, then diluted 16-fold three times with the assay medium, then diluted 4-fold seven times, and then loaded with eight dilution gradients to prepare two duplicate wells. 3T3 cells were cultured and passaged in complete medium. 3T3 cells in the logarithmic growth phase were thoroughly digested with pancreatin to form single cells, collected, and diluted to a cell density of 1.0 x 10 in the assay medium. 5 The cell suspension was adjusted to approximately 10 cells / ml and a cell suspension was prepared. The cell suspension was then inoculated into a 96-well plate containing the sample, with 50 μl per well. The plate was then incubated for 66-72 hours in a 37°C incubator containing 5% CO2. 10 μl of MTT solution was added per well and the plate was then incubated for 4-6 hours in a 37°C incubator containing 5% CO2. 100 μl of 10% SDS was added per well and the plate was then incubated overnight in a 37°C incubator containing 5% CO2. Colorimetry was performed using a microplate reader, with the OD values ​​read and recorded at wavelengths of 570 nm or 570 nm / 630 nm. The corresponding OD-rhAFGF concentration curve was then plotted, and its activity was calculated using the following formula:

[0040] Biological activity of sample (U / ml) = Pr × Ds × Es / (Dr × Er) Pr is the biological activity of the preparation in U / ml, Ds is the predilution factor of the test substance, Dr is the pre-dilution factor of the standard, Es is the dilution factor of the test substance corresponding to the half-effective dose of the standard, Er is the dilution factor of the standard at half the effective dose.

[0041] Table 1: Relative activity determined from stability at 25°C (relative activity on day 0 is set to 100%) [Table 1]

[0042] Table 2: Relative activity determined from stability at 40°C (relative activity on day 0 is set to 100%) [Table 2]

[0043] Based on the above, it cannot be recognized that the specific implementation of the present invention is limited to these descriptions, and various simple simulations or substitutions that a person skilled in the art can make without departing from the concept of the present invention should all be considered to fall within the scope of protection of the present invention as defined by the appended claims.

Claims

1. comprising a gel matrix, a thickening agent, and a protective agent; the gel matrix is ​​one or more selected from the group consisting of sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, sodium polypropylene, sodium hyaluronate, polyethylene glycol, and gelatin; The thickener is polyvinyl alcohol, the protective agent is selected from a combination of histidine and low molecular weight sodium heparin; Preferably, the average molecular weight of the low-molecular-weight sodium heparin is 3,000 to 8,000.

2. 2. The recombinant human acidic fibroblast growth factor (aFGF) gel of claim 1, wherein the mass of the gel matrix is ​​0.5-5.0%, preferably 1.0-3.0%, of the mass of the gel.

3. 2. The recombinant human acidic fibroblast growth factor (aFGF) gel of claim 1, wherein the mass of the thickener is 0.5-3.0%, preferably 1-2%, of the mass of the gel.

4. 2. The recombinant human acidic fibroblast growth factor (aFGF) gel according to claim 1, wherein the mass of the histidine is 0.8 to 2% of the mass of the gel, preferably 1 to 1.6% of the mass of the gel, and the mass of the low molecular weight sodium heparin is 0.05 to 1% of the mass of the gel, preferably 0.08 to 0.2% of the mass of the gel.

5. 2. The recombinant human acidic fibroblast growth factor (aFGF) gel according to claim 1, further comprising a buffer solution, wherein the buffer solution is selected from a phosphate buffer system, a citrate buffer system, and a Tris buffer system, preferably a phosphate buffer system, and the buffer solution preferably has a pH of 6.0-7.0 and a concentration of 0.01-0.05 mol / L, preferably 0.02 mol / L.

6. The relative amount of recombinant human acidic fibroblast growth factor in the gel is 2-8×10 ３ 2. The recombinant human acidic fibroblast growth factor (aFGF) gel of claim 1, wherein the amount of the recombinant human acidic fibroblast growth factor (aFGF) is 1.1 IU / g.

7. 2. The recombinant human acidic fibroblast growth factor (aFGF) gel of claim 1, further comprising a moisturizer and a preservative.

8. 7. The recombinant human acidic fibroblast growth factor (aFGF) gel according to claim 6, wherein the moisturizing agent is selected from glycerol and the preservative is selected from parahydroxybenzoic acid ester preservatives.

9. 7. The recombinant human acidic fibroblast growth factor (aFGF) gel according to claim 6, wherein the preservative is one or more selected from the group consisting of methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and isopropyl parahydroxybenzoate.

10. 7. The recombinant human acidic fibroblast growth factor (aFGF) gel according to claim 6, wherein the mass of the preservative is 0.1-3% of the mass of the gel, preferably 0.2-1% of the mass of the gel, and the mass of the moisturizer is 0.2-4% of the mass of the gel, preferably 0.5-2% of the mass of the gel.

Images